Embryonic stem cells are characterized as cells with high telomerase activity, infinite capacity for self-renewal, and pluripotency (the ability to differentiate into any cell type represented in the three germ layers, ectoderm, endoderm and mesoderm). As a result, a great deal of hope is associated with the potential application of human embryonic stem (hES) cells in the fields of gene/cell therapy and regenerative medicine.
The first successful derivation of human embryonic stem (hES) cells was reported by Thomson, J. A. et al. (Science (1998) 282:1145-1147). In that study, hES cells were isolated from the inner cell mass of blastocysts and plated onto mitotically inactivated murine embryonic fibroblast (MEF) feeder cells. Initial hES cell derivation and culture has originated from culturing practices developed predominantly for murine ES (mES) cells over the past 25 years. While MEF feeders have proved to be a robust surface for long term cultures of hES cells, this methodology has several limitations. First, there are concerns about potential animal pathogens that could be transferred to the hES cells, making them unsuitable for clinical applications. Second, it is inconvenient and tedious to grow and maintain two cells types. Furthermore, it is difficult to transfect and genetically manipulate the compacted hES cell colonies on feeders. For all these reasons, efforts have been initiated to develop feeder-free conditions for culturing hES cells.
Mouse ES cell research has served as a foundation for furthering our understanding of the mechanism of regulating self-renewal of hES cells. Manipulation of specific signaling regulators has facilitated the development of feeder-free conditions required for propagating undifferentiated hES cells. Recent studies have clearly delineated that hES cells can be grown in the absence of feeders, as long as they are plated on an appropriate extra-cellular matrix (ECM) and cultured in media either conditioned on feeder cells or supplemented with soluble factors.
An extracellular matrix (ECM) is a three-dimensional molecular complex that varies in composition, and may include components such as laminin, fibronectin, collagens and other glycoproteins, hyaluronic acid, proteoglycans, and elastins. Examples of commercially available extracellular matrices include, for example, ECM gel by Sigma-Aldrich or Matrigel™ Matrix by Becton, Dickinson & Company. Matrigel™ Matrix is a reconstituted basement membrane isolated from mouse Engelbreth-Holm-Swarm (EHS) tumor. Matrigel™ matrix is composed predominantly of laminin, collagen IV, entactin and heparan sulfate proteoglycans. Matrigel™ matrix also contains a number of growth factors, such as EGF, IGF-1, PDGF, TGF-β, VEGF and bFGF.
Prior to the present invention, it was known that hES cells could be propagated in an undifferentiated state on an ECM surface using conditioned media from different types of feeder cells. There are a number of advantages in using an ECM as a feeder-free substrate for culturing hES cells. For instance, DNA, RNA and protein isolation is easier on an ECM surface due to lack of potential contamination from the feeder cells. In addition, genetic manipulation of hES cells requires efficient transfection capabilities. Transfection efficiencies of hES cells grown on a Matrigel surface are improved compared to those grown on feeders. When cultured on BD Matrigel™ Matrix, it was previously shown that hES cells form “monolayer-like” colonies, making individual cells more accessible for penetration with DNA or SiRNA. Although other ECM proteins have also been successfully used for feeder-free cultures, several prior studies have suggested that a combination of ECM proteins is required to perform as well as Matrigel™ Moreover, purified proteins are generally more expensive and not as well tested for long-term performance as Matrigel™.
Despite the wide acceptance of Matrigel™ as a viable substrate for long term feeder-free culture of hES cells, it has one key limitation. Since Matrigel™ is derived from the EHS tumor, lot-to-lot variability in protein concentration and composition is inherent. In order to limit the amount of time researchers spend today pre-screening lots of Matrigel™ and coating substrates on a weekly basis, it would be of benefit to provide a stable, ready-to-use substrate coated with an optimal ECM composition for ES culture. Preferably, this culturing system would provide lot-to-lot consistency, which is needed for standardized ES cultures.